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Basic toxicokinetics

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basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication/study report which meets basic scientific principles

Data source

Referenceopen allclose all

Reference Type:
NTP Technical Report on the Toxicology and Carcinogenesis Studies of alpha-Methylstyrene (CAS NO. 98-83-9) in F344/N rats and B6C3F1 mice (inhalation studies)
Bibliographic source:
NTP Technical Report 543. National Toxicology Program, P.O. Box 12233, Research Triangle Park, NC 27709, 216 pp.
Reference Type:
Metabolism and disposition of alpha-methylstyrene in rats
De Costa KS, Black SR, Thomas BF, Burgess JP & Mathews JM
Bibliographic source:
Drug Metab Dispos 29, 166 – 171

Materials and methods

Objective of study:
Test guideline
no guideline followed
not applicable
Principles of method if other than guideline:
The toxicokinetics of alpha-methylstyrene were extensively investigated in rats with oral, intravenous and inhalative exposure:
i.v.: 10 mg/kg
oral: 1000 mg/kg
inhalation: 300 or 900 ppm over 6 hrs (nose-only).

Detailed information is given in the field "Details on study design"
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Nonradiolabeled alpha-methylstyrene (lot #0922OLG) was purchased from the Aldrich Chemical Company with a stated purity of >= 99 %. Radiolabeled alpha-methylstyrene (lot #950612), randomly radiolabeled with 14C in the phenyl ring, was received from Wizard Laboratories, Inc., at a specific activity of 1.0 mCi/mmol. [14C]alpha-methylstyrene was initially determined to be approximately 99 % radiochemically pure by HPLC. Purity of [14C]alpha-methylstyrene in dose formulations was reestablished at >= 98 % during the studies.

Test animals

Fischer 344
Details on test animals or test system and environmental conditions:
Adult male F344/N rats were purchased from Charles River Laboratories, Inc. Animals were quarantined at least 1 week before they were used in a study. Rats were fed Certified Purina Rodent Chow #5002 and were furnished tap water ad libitum. Rats were housed in standard polycarbonate cages and moved to individual glass metabolism chambers the day before study start for acclimation. These chambers provided for separate collection of urine, faeces, and breath components for the oral and intravenous exposures. For all studies, rats were sacrificed by an intracardiac injection of sodium pentobarbital.

Administration / exposure

Route of administration:
other: intraveneous, inhalation and oral dosing via gavage
Duration and frequency of treatment / exposure:
single dosing
Doses / concentrations
Doses / Concentrations:
i.v.: 10 mg/kg
oral: 1000 mg/kg
inhalation: 300 or 900 ppm over 6 hrs (nose-only)
No. of animals per sex per dose / concentration:
1 - 4
Control animals:
Details on study design:
For nose-only exposures, Battelle restrainers were used during the exposure phase of the inhalation study. Animals were acclimated to the restrainers for 2 hours each day for 2 days prior to exposure. Restrainers afforded separate collection of urine and faeces excreted during exposure. Upon termination of the 6-hour exposure and removal from restrainers, a stream of warm air was briefly directed over each rat to remove [14C]alpha-methylstyrene deposited on the fur and to prevent ingestion of [14C]alpha-methylstyrene through grooming. Rats were then immediately transferred to glass metabolism chambers.
An indwelling jugular cannula was surgically implanted into animals used in the inhalation study to facilitate serial collection of blood samples. Animals were allowed to recover approximately 24 hours prior to exposure.
Intravenous dose formulations contained 13.3 to 23.6 uCi [14C]alpha-methylstyrene and an appropriate amount of Emulphor EL-620 and phosphate buffered saline (1:20, respectively) in a dose volume of 1 mL/kg. Oral dose formulations contained 43.8 uCi [14C]alpha-methylstyrene, an appropriate amount of nonradiolabeled alpha-methylstyrene, and sufficient corn oil for a dose volume of 5 mL/kg. Alpha-methylstyrene used for generation of atmospheres during nose-only inhalation exposures contained appropriate masses of radiolabeled and nonradiolabeled alpha-methylstyrene to deliver approximately 20 uCi per rat at 300 or 900 ppm.
Intravenous doses (10.8 mg alpha-methylstyrene/kg bw) were drawn into a syringe equipped with a Teflon-tipped plunger and a 27-gauge hypodermic needle. Excess dose formulation was wiped off the needle before weighing the filled dosing apparatus. Intravenous doses were injected into a lateral tail vein. After dosing, the needle was wiped clean with a Kimwipe, and the empty dosing apparatus was reweighed. The Kimwipe was placed into a vial containing 2 mL methanol and analyzed by LSS. Each dose was calculated as the difference between the weights of the filled and empty dosing apparatus, less the amount found in the wipes. To determine the concentration of [14C]alpha-methylstyrene in the dose formulation, two weighed aliquots were taken before, two after, and one in the midst of dosing a series of animals. These aliquots were assayed for 14C using LSS.
The oral dose (1000 mg/kg) was administered to one rat by intragastric gavage. The dose, contained in a 2.5-mL syringe fitted with a Teflon-tipped plunger and a gavage needle (16-gauge ball-tipped), was weighed after excess dose was removed from the outside with a Kimwipe. The wipe was placed into a scintillation vial containing 2 mL of methanol and analyzed by LSS after addition of fluor. The determination of actual dose delivered was performed as for the intravenous study.
For generation and delivery of alpha-methylstyrene vapour to animals during 6-hour, nose-only inhalation exposures, conditioned room air was pumped into the inlet stream using a Gast Model 0531 rotary, vane type pump. Mass flow controllers regulated total inlet and outlet flow to deliver a constant flow (400 mL/minute) of freshly generated vapour to each nose port while removing this flow from the ports at a slightly lower rate. This resulted in a slightly positive pressure in the apparatus (1 to 2 inches H2O, Dwyer Magnehelic pressure gauge). Liquid alpha-methylstyrene was metered from a syringe (Harvard Apparatus Model 22 infusion pump) in the generation column. The appropriate infusion rate was determined experimentally during preliminary trial runs. The column was wrapped with a specially designed heating mantle to maintain a temperature of 155 +/- 5 degrees C, sufficient for vapour generation. Alpha-methylstyrene vapour from the generation column entered a glass mixing chamber (600 mm × 11 mm interior diameter) prior to passing through a 0.5 u PTFE membrane filter and into the inlet manifold. The inlet manifold equally divided total flow into 400 mL/minute portions delivered to each nose port through equal lengths of stainless steel tubing. Noninhaled and expired vapour exited the nose ports through equal lengths of copper tubing passing into an outlet manifold where the portions were recombined into a single air stream. This single air stream then passed through eight vapour scrubbing traps filled with acetonitrile to remove residual radiolabel prior to exiting via another Gast vacuum pump to the laboratory fume hoods. The first four scrubbing traps were cooled to 0 degrees C, and the remaining four traps were cooled to 60 degrees C with a dry ice/isopropanol slurry. A constant 30 mL/minute flow of alpha-methylstyrene vapour derived from the inlet flow stream just prior to the inlet manifold was continuously directed through 1/8 inch outside diameter stainless steel tubing into a series of gas sampling valves and a 1 mL sample loop before venting to the laboratory fume hood. Actuation of the gas sampling valve transferred the sample loop contents into the carrier gas stream of the Varian 3700 gas chromatograph, initiating analysis of the 1 mL vapour sample.
Alpha-methylstyrene vapour concentration in exposure atmospheres was quantitated by an automated gas chromatographic system.
Absorbed doses (mg alpha-methylstyrene/kg bw) in the inhalation studies were calculated using the total mass (mg) of alpha-methylstyrene absorbed per animal during exposure. These mass values were determined by dividing the total 14C (uCi) recovered in tissues, residual carcass, volatile breath, and excreta by the specific activity (uCi/mg) of the solution used for alpha-methylstyrene vapour generation (four animals per study). Total masses of alpha-methylstyrene absorbed by animals sacrificed immediately following cessation of exposure (three animals per study) were calculated as noted above except no excreta were collected from this group during or after exposure. Exhaled volatile components eliminated in the short interval between cessation of exposure and sacrifice were also not accounted for in this group, thereby precipitating their exclusion in the calculation of the mean absorbed dose reported per study. The mean mass of alpha-methylstyrene absorbed by four rats per study used for excreta collection was used to estimate the absorbed dose (mg/kg) for the five rats per study used for blood collection.
Urine and faeces were collected separately into round-bottom flasks, cooled with dry ice, and removed at 6, 12 (urine only), 24, 48, and 72 hours postdosing in the intravenous study and postinitiation of exposure in the inhalation studies. Urine and faeces were collected at 6, 24, and 48 hours postdosing in the oral study. Residual bladder urine was removed at sacrifice and combined with the 72-hour collection. In the inhalation studies, 6-hour samples contained all urine and faeces excreted into restrainers during exposure. During exposure, urine and faeces excreted into restrainers were immediately transferred to scintillation vials. Samples were stored in the dark at approximately –20 degrees C until analyzed.
Expired volatile compounds and carbon dioxide (intravenous study only) were collected by passing room air through the metabolism chambers and a series of traps. The first trap was filled with 40 to 75 mL absolute ethanol and cooled to 0 degrees C in an ice/water bath. The second trap, also containing absolute ethanol, was cooled to –60 degrees C using a dry ice/isopropanol slurry. The third and fourth traps (intravenous study only) were filled with approximately 500 mL of 1 N NaOH. Traps were changed at 6, 12, 24, 48, and 72 hours postdose and 12, 24, 48 (900 ppm only), and 72 hours (900 ppm only) postinitiation of dose during the intravenous and inhalation studies, respectively.
Serial blood samples (300 uL each) were collected through the jugular cannulae from five animals for 24 hours, beginning 5 hours after initiation of exposure, in the 300 and 900 ppm inhalation studies. Immediately following collection, two approximately 75 uL aliquots were transferred to scintillation vials filled with 2 mL of Soluene-350 for 14C quantitation. The remaining approximately 150 uL was transferred to a microcentrifuge tube for extraction according to the method described above.
Adipose tissues (two samples), muscle (two samples), skin (ears), as well as entire kidney, liver, spleen, lung, testes, bladder, heart, and brain were removed and assayed for 14C content. Additionally, stomach, small intestine, caecum, and large intestine were removed and assayed for 14C content. No tissues were excised in the oral or pilot intravenous pharmacokinetic studies. During the inhalation studies, skin samples were removed from the hind leg instead of the ears.
Aliquots of urine, ethanol, and NaOH were added directly to vials containing scintillation cocktail. Samples of tissue, faeces, and blood (0.1 to 0.3 g) were digested in Soluene-350 (2 mL). After digestion, samples requiring bleaching were decolorized with perchloric acid/hydrogen peroxide prior to addition of scintillation cocktail. Stomach, small intestine, caecum, large intestine, and the carcass were digested in 2 N ethanolic sodium hydroxide, and aliquots were added to scintillation cocktail for analysis by LSS.
Samples from each urinary collection interval up to 48 hours from one rat (10 mg/kg intravenous) were analyzed by HPLC. Analysis of the urine by HPLC identified six metabolites: A (unknown), B (2-phenyl-1,2-propanediol glucuronide), C (2-phenyl-1,2-propanediol), D (atrolactic acid), E [S-(2 hydroxy-2-phenylpropyl)-N-acetylcysteine], and F (2-phenyl propionic acid). For the inhalation studies, urine from a different rat for each collection interval between 6 and 48 hours was analyzed. The urine was filtered through a Millex HV 0.45 um filter. Radiochemical concentrations of the samples were determined by LSS. Profiles of 14C-labeled components were determined by HPLC as described above.
After the urinary metabolite profiles had been established in the intravenous study, attempts were made to determine which metabolites were present as glucuronide, sulfate, or other conjugates. Aliquots of urine from the intravenous study were incubated with beta-glucuronidase, sulfatase, or acylase. To a vial containing 1000 units of beta-glucuronidase was added 420 uL of deionized distilled water; 200 uL of this solution was boiled for 10 minutes and then flash frozen to deactivate the enzyme. 200uL of either the heat deactivated enzyme or the active enzyme was added to 50 uL of urine from the 0 through 6 and 6 through 12 hour collections and to 100 uL of urine from the 12 through 24 and 24 through 48 hour collections. The mixtures were then incubated for 1 hour at 37 degrees C. Sulfatase incubations contained 50 uL of urine (0 through 6 or 12 through 24 hour collection), 250 uL of Trizma buffer (pH 7.6), and 250 uL of sulfatase solution (10 to 20 units/mL). Controls were prepared with heat deactivated and flash frozen enzyme. The incubations were heated at 37 degrees C for 1 hour. Alternately, urine samples (100 uL) were incubated at 37 degrees C for 6 hours with 100 uL acylase solution [2 mg/mL containing 7000 units/mg in 0.9 M potassium phosphate buffer (pH 7.4)]. These incubation mixtures were chromatographed as described previously, and the profiles compared with those from untreated urine. There was no noticeable change in the profile following treatment with sulfatase or acylase. However, following treatment with beta-glucuronidase, metabolite B was transferred to a compound that coeluted with metabolite C.
Urine collected 6 to 24 hours postdosing from a rat administered alpha-methylstyrene by oral gavage (1000 mg/kg) was used for isolation and purification of metabolites using the HPLC method described previously. Metabolites were manually collected from the radioactivity detector following multiple runs. Eluant containing a given metabolite was pooled; the organic solvent was removed by rotary evaporation; and the residue (except for metabolite D) was brought to dryness by lyophilization. With metabolite D, the residue was basified to pH 10 with 1 N NaOH prior to lyophilization, and the resulting residue was reconstituted in methanol. A portion of this methanolic solution was reanalyzed by HPLC to confirm that the isolation procedures caused no degradation of the metabolite, and the remainder of the solution was dried under a stream of nitrogen. Metabolites C, D, E, and F were further purified by use of a Waters C18 Sep-Pak Plus extraction column prior to analysis by gas chromatography-mass spectrometry. Metabolite E was rechromatographed to eliminate contamination with metabolite D using the method described above for the initial isolation; then metabolite E was incubated with acylase. Metabolite B was treated with beta-glucuronidase and rechromatographed using the same HPLC conditions. A new peak appeared that coeluted with metabolite C, and this analyte was collected and concentrated to dryness. The trimethylsilane derivatives of metabolite C, the aglycone of metabolite B, a standard of 2-phenyl-1,2-propanediol, metabolite D, and a standard of atrolactic acid were prepared and analyzed by GC/MS. A standard of 2-phenylpropionic acid and metabolite F were analyzed by GC/MS. A sample of metabolite B was further purified using a Microsorb-MV phenyl analytical column with an isocratic mobile phase of 10 % acetonitrile in aqueous 1 % acetic acid, with a flow rate of 1 mL/minute. This sample of metabolite B was analyzed by 1H- and 13C-Distortionless Enhanced Polarization Transfer (DEPT) NMR. Metabolite E was analyzed by 1H- and 13C-NMR, and its trimethylsilane derivative was analyzed by GC/MS.
Aliquots (150 uL) of blood from the inhalation studies were extracted with 300 uL of acetonitrile and then centrifuged at 14000 or 16000 g; the supernatants were transferred to 0.5 dram vials. Nonradiolabeled alpha-methylstyrene, atrolactic acid, and 2-phenylpropionic acid standards were added to each vial. Immediately prior to analysis by HPLC, 300 uL of water was added to each extract. Whole blood obtained from rats immediately after 6-hour nose-only inhalation exposure (900 ppm) was centrifuged to obtain red blood cells. Red blood cells were washed with 0.9 % saline, lysed with distilled deionized water, and centrifuged. A 20 uL aliquot of supernatant was diluted with 300 uL of the mobile phase (initial conditions) used for analysis by HPLC. To determine whether bound radiolabel was associated with haeme, an HPLC method that separates haeme from globin was used.
Blood alpha-methylstyrene concentration versus time data obtained during and postexposure in the 300 and 900 ppm inhalation studies were analyzed by noncompartmental and compartmental techniques using WinNonlin software. Noncompartmental analysis of data from individual animals was performed with WinNonlin Model 202 for infusion, where the inhaled dose was used as the infused dose.
Data sets from individual animals were also analyzed by compartmental techniques. The data were initially fit to a two-compartment model with zero-order absorption and first-order elimination (WinNonlin Model 9). Two additional two-compartment models were written to simultaneously solve pooled data from all animals within an exposure concentration from each of the two inhalation experiments. Each of these models permitted zero-order absorption. The models differed in that the first model contained a description of first-order elimination and the second contained a description of nonlinear (Michaelis-Menten) elimination. All pharmacokinetic analyses were conducted on weighted data (1/YHAT, where YHAT is the predicted alpha-methylstyrene concentration). For models in which data from two exposure concentrations were solved simultaneously, the dose rate (mg/kg per hour, calculated as dose received in mg/kg divided by the duration of exposure in hours) was required as model input.
Not further specified

Results and discussion

Metabolite characterisation studies

Metabolites identified:

Any other information on results incl. tables

Intravenous Study for Urinary Metabolite Identification

Intravenous doses of alpha-methylstyrene (10 mg/kg) were mainly excreted in the urine with 76 +/- 2 % excreted in the first 24 hours postdosing and 86 +/- 1 % by 72 hours. Faecal elimination accounted for 2 % of the dose. Exhalation of volatile organics and carbon dioxide accounted for only 2 % and 0.02 % of the dose, respectively. Concentrations of alpha-methylstyrene equivalents were low, and only 0.3 % of the radioactivity was recovered in the tissues. The concentration of alpha-methylstyrene and/or its metabolites in blood was 16 ng Eq per gram.

The profiles of metabolites present following an intravenous dose of alpha-methylstyrene (10 mg/kg) were determined for each urinary collection interval up to 48 hours postdosing for one rat. The peak eluting at 10:25 (metabolite D) coelutes with atrolactic acid, and over 20 % of the administered dose was excreted as this metabolite. Metabolite E was most abundant in the early urine collection (0 to 6 hours), with considerably less detected in later collections, suggesting that it may be an early intermediate metabolite of alpha-methylstyrene.

Oral Study for Urinary Metabolite Identification

Alpha-methylstyrene was administered orally to one rat at a dose of 1000 mg/kg in an effort to obtain as much urinary metabolite as possible. Urine from this experiment was chromatographed and gave a similar profile as the urine from the intravenous study. Five of the metabolite peaks were identified by GC/MS. Metabolite B was treated with beta-glucuronidase, and the aglycone was analyzed by GC/MS. The spectrum of the bis-trimethylsilane derivative of a 2-phenyl-1,2 -propanediol standard had no M+ at 296 but had peaks at m/z 281 (loss of a methyl), 193 (loss of CH2O-trimethylsilane ), and 147. The spectra of metabolite C and the aglycone of metabolite B were virtually identical to that of the bis-trimethylsilane derivative of a 2-phenyl-1,2-propanediol standard. Therefore, it was concluded that metabolite B is a glucuronide of 2-phenyl-1,2-propanediol and that metabolite C is 2-phenyl-1,2-propanediol.

Metabolite B was analyzed by 13C-DEPT NMR in an effort to determine the position of attachment of the glucuronide group in the conjugate. Of the two oxygenated carbon atoms, only the terminal methylene group had protons with which the pulsed carbon nucleus could relax. Therefore, the position of the methylene resonance was determined in these characterization experiments using NMR. The spectra displayed a doublet for each carbon bearing a proton, possibly indicating that this metabolite was present as a pair of diasteriomers. The 13C-DEPT NMR spectrum of 2-phenyl-1,2-propanediol was determined in order to interpret the spectra for metabolite B, and the methylene resonance appeared at 72 ppm. If the glucuronide conjugate were attached to the carbon alpha to the phenyl ring, the chemical shift of the methylene carbon would have been approximately 3 ppm upfield (at 69 ppm) relative to that for the unconjugated 2-phenyl-1,2-propanediol; had it been at the beta carbon, the shift would have been approximately 10 ppm downfield (at 82 ppm). In the 13C-DEPT NMR spectrum of metabolite B, the methylene resonances were at 79 ppm, consistent with the attachment of the glucuronide moiety to the carbon beta to the phenyl ring.

A bis-trimethylsilane derivative of an atrolactic acid standard and metabolite D were analyzed by GC/MS, and the spectra were identical. A standard of 2 -phenylpropionic acid and metabolite F were analyzed by GC/MS, and both spectra contained a molecular ion at m/z 150 and signals at m/z 105 (loss of COOH) and 77. Metabolite E was treated with acylase, and the incubation mixture was chromatographed as described previously for isolation of the peak. The profile was compared with the HPLC profile of the untreated urine. Treatment with acylase converted metabolite E to a new component that eluted between metabolites A and B. The 1H NMR spectrum of metabolite E was consistent with a N-acetylcysteine conjugate resulting from reaction of glutathione with the epoxide of alpha-methylstyrene, followed by further metabolism to the mercapturate. The 13C-DEPT NMR also corroborated this finding. The 13C chemical shift of the methylene group beta to the ring was consistent with attachment of the sulphur atom to this carbon atom. Trimethylsilane derivatives of the mercapturate(s) were prepared for analysis by GC/MS. Two di-derivatized and two tri-derivatized products were present in roughly equal amounts. The fragmentation patterns (in particular the ion at 193 a.m.u.) in these spectra were also consistent with the formation of just one of two possible positional isomers for the mercapturate. The NMR and mass spectral data indicated that a diasteriomeric pair of mercapturates was formed as metabolites.

The presence of roughly equal amounts of the diasteriomeric mercapturates suggests that the initial epoxidation of alpha-methylstyrene is not sterioselective and proceeds with no marked preference for the antarafacial or suprafacial addition of active oxygen to yield enantiomeric epoxides. Both enzymatic hydrolysis and glutathione conjugation of epoxides are known to proceed by SN2 reactions. Therefore, enzymatic hydrolysis can yield enantiomeric diols. Further oxidation of the terminal hydroxy group of these diols to form atrolactic acid does not affect the chiral centre at the benzyl position, and the potential products are enantiomers. However, conjugation with a chiral molecule such as glutathione or glucuronic acid would produce diasteriomeric metabolites from the enantiomeric products, as is the case with the mercapturates and glucuronides characterized in these studies.

Inhalation Study

The time weighted average alpha-methylstyrene vapour concentrations during exposure for the 300 and 900 ppm groups were 304 and 900 ppm. Animals exposed to 300 or 900 ppm received mean doses of 130.8 and 340.1 mg/kg, or 26.63 and 20.05 uCi, respectively.

For the 300 ppm group, urinary excretion was the main route of elimination, comprising 88.2 % of the absorbed dose, with volatile breath and faeces accounting for 3.1 and 2.2 %, respectively. These trends were consistent for all collection intervals. Over 90 % of the absorbed dose was eliminated within 48 hours post-initiation of exposure. The same pattern of elimination was observed in the 900 ppm group; 92.4 % was excreted in urine, with volatile breath and faeces accounting for 2.5 and 2.6 %, respectively.

Tissue distribution of radioactivity at 6, 24 (300 ppm only), and 72 hrs after initiation of inhalation: 2.6 – 10.1 % and 1.1 – 2.4 % of the inhaled radiolabeled alpha-methylstyrene was recovered in the residual carcass and tissues in the 300 and 900 ppm groups, respectively. The highest concentrations of radiolabeled alpha-methylstyrene (ug Eq/g tissue) were observed in adipose, bladder, liver, kidney, and skin at 6, 24 (300 ppm only), and 72 hours after initiation of exposure. This is consistent with the lipophilic nature of alpha-methylstyrene and the fact that most of the dose was eliminated in urine. Elevated levels of radiolabel were present in the small intestine compared to the stomach and large intestine, suggesting biliary excretion and re-absorption of [14C]alpha-methylstyrene-derived metabolite(s). Tissues for the 24-hour time point in the 300 ppm group were removed from animals used for serial blood sampling.

The metabolite profile observed in urine from 300 and 900 ppm male rats was qualitatively the same as that in the intravenous study. Much more atrolactic acid was observed in urine collected during the first 24 hrs in the inhalation studies compared to the intravenous study. Rats exposed to 900 ppm exhibited nearly a twofold increase in excretion of atrolactic acid, accompanied by a corresponding drop in excretion of 2-phenyl-1,2-propanediol glucuronide, in urine collected between 12 and 24 hrs compared to rats exposed to 300 ppm.

Characterization of the 14C Profile in Blood

In extraction method development experiments, recovery of carbon-14 from blood spiked with [14C]alpha-methylstyrene was 95 +/- 6 %. Recoveries of carbon-14 from blood samples obtained from the 300 and 900 ppm groups were 74 +/- 10 % and 82 +/- 9 %, respectively. These recoveries suggest sequestration of metabolites by red blood cells. Chromatographic analysis of red blood cell lysate showed that no radioactivity was associated with heme.

Alpha-methylstyrene concentrations in blood dropped precipitously in the 300 ppm rats upon cessation of exposure, from more than 6 ug/mL just prior to termination of exposure (5.5 hrs) to an average of 0.97 ug/mL at 7 hrs. From 7 – 24 hrs, alpha-methylstyrene concentrations in blood decreased at a much slower rate. Alpha-methylstyrene concentration in blood dropped from more than 24 ug/mL at 5.5 hrs into the exposure to ca. 10 ug/mL in the first hour after cessation of exposure in the 900 ppm rats.

Four metabolites were extracted from blood obtained in the inhalation study. The major component at all time points was 2-phenyl-1,2-propanediol. Atrolactic acid, 2 -phenylpropionic acid, and an additional radiolabeled component noted as blood metabolite 1 were also observed. Identities of atrolactic acid, 2-phenyl-1,2 -propanediol, 2-phenylpropionic acid, and alpha-methylstyrene peaks were established by co-elution with non-radiolabeled standards.

Applicant's summary and conclusion

Interpretation of results (migrated information): no bioaccumulation potential based on study results
Alpha-methylstyrene was readily metabolized by rats to form products that were chiefly excreted in the urine. There was little accumulation of radiolabeled equivalents in the tissues.
Executive summary:

Male F344/N rats were exposed to alpha-methylstyrene via intravenous or nose-only inhalation exposure. In both studies, the substance was eliminated primarily in the urine (approximately 90 %) within 72 hours, with volatile breath and feces accounting for only a small amount (1 - 3 %) of elimination. In the inhalation study, the elimination half-life was calculated at 3 to 5 hours, with the highest concentrations of alpha-methylstyrene-derived radioactivity retained in the adipose tissue, urinary bladder, liver, kidney, and skin. Following intravenous dosing, the kidney, heart, lung, liver, urinary bladder, and spleen retained the highest concentrations of radioactivity. In both the intravenous study and the inhalation study, the major urinary metabolites of alpha-methylstyrene were the glucuronide conjugate of 2 -phenyl-1,2-propanediol and atrolactic acid. In the inhalation study, the major metabolites in the blood were 2-phenyl-1,2-propanediol and 2-phenylpropionic acid.

Based on these studies, the proposed metabolic pathway for alpha-methylstyrene involves an initial non-stereoselective epoxidation followed by hydrolysis to form 2-phenyl-1,2-propanediol followed by either oxidation to atrolactic acid or formation of the glucuronide conjugate, conjugation with glutathione and subsequent cleavage to the mercapturate, or rearrangement to form an aldehyde that is oxidized to yield 2-phenylpropionic acid. The dose-dependent pharmacokinetic parameters coupled with decreased excretion of 2-phenyl-1,2-propanediol glucuronide at 900 ppm indicate that glucuronide formation was saturated at this dose.